Recombinant production of human and bovine receptors for modified low-density lipoprotein

ABSTRACT

cDNAs encoding the human and bovine vascular endothelial receptors for modified low-density lipoprotein are provided. Also described are corresponding expression vectors, their use in the recombinant production of the encoded receptors, and the receptor polypeptides obtained thereby.

FIELD OF THE INVENTION

The present invention relates to a mammalian receptor for modifiedlow-density lipoprotein (LDL), and in more detail relates to a mammalianvascular endothelial receptor for modified low-density lipoprotein.

BACKGROUND OF THE INVENTION

Vascular endothelial dysfunction has been pointed out as an importantindex in the early stages of progressive atherosclerosis. The vascularendothelial cell releases many sorts of humoral factors to keepcirculatory homeostasis. The vascular endothelial function is inhibitedby physical stimuli or various substances including the most importantfactor, oxidized low-density lipoprotein, which is a kind of modifiedlow-density lipoproteins. For example, the vascular endothelial cellreleases nitrogen monoxide as a vasohypotonic factor to adjust vasculartonus. The release of nitrogen monoxide is inhibited by the oxidizedlow-density lipoprotein.

It has been known that macrophages or vascular endothelial cellsinternalize the modified low-density lipoprotein through a receptorother than receptors for low-density lipoprotein. The macrophagesinternalize the modified low-density lipoprotein through scavengerreceptors, which have already been structurally analyzed (cf., PCTPatent Japanese Publication Nos. 6(1994)-500765 and 6(1994)-508604, andJapanese Patent Provisional Publication No. 3(1991)-290184). Themacrophages are then changed to foam cells, which are specific inarteriosclerotic focus. Since the macrophage scavenger receptors are notfound in vascular endothelial cells, it has been anticipated thatreceptors of another structure are present in the vascular endothelialcells (cf., Hidenori Arai, Toru Kita, Oxidized LDL, Metabolism 28/4,1991).

For the reasons mentioned above, it is necessary to analyze thestructure of an endothelial receptor for modified low-densitylipoprotein, namely the amino acid sequence of the receptor. However,the structure and the amino acid sequence have not yet been elucidated.

DISCLOSURE OF THE INVENTION

According to study of the present inventors, the structure of thevascular endothelial receptor for modified low-density lipoprotein isnow elucidated. The amino acid sequences of the vascular endothelialreceptor for modified low-density lipoprotein are set forth in SEQ IDNOS: 2, 4 and 6.

There is provided by the present invention a DNA sequence essentiallyencoding a mammalian vascular endothelial receptor for modifiedlow-density lipoprotein.

The DNA sequence can be cDNA clones derived from the open reading frameof a gene corresponding to a native mammalian vascular endothelialreceptor for modified low-density lipoprotein. The DNA sequence can alsobe a sequence which is capable of hybridization to the above-mentionedcDNA clones and encodes a biologically active mammalian vascularendothelial receptor for modified low-density lipoprotein. Further, thesequence can be degenerate as a result of the genetic code to theabove-mentioned DNA sequences. The degenerate encodes the samebiologically active receptor for modified low-density lipoprotein.Therefore, the present invention provides a DNA sequence set forth inSEQ ID NO: 1, 3 or 5(DNA having the sequence) or an analogue thereof,which corresponds to the region encoding a mammalian vascularendothelial receptor for modified low-density lipoprotein.

The present invention also provides a CDNA clone having a DNA sequenceset forth in SEQ ID NO: 1, 3 or 5 or an analogue thereof, whichcorresponds to the region encoding a mammalian vascular endothelialreceptor for modified low-density lipoprotein.

The present invention further provides a DNA sequence which is capableof hybridization to a cDNA clone of a DNA sequence set forth in SEQ IDNO: 1, 3 or 5 (DNA having the sequence) in 20% (v/v) formamide at 42°C., and encodes a protein of a mammalian vascular endothelial cell, saidprotein having a function of binding a modified low-density lipoprotein(namely a receptor thereof).

The present invention furthermore provides a DNA sequence which isdegenerate as a result of the genetic code to a DNA sequence set forthin SEQ ID NO: 1, 3 or 5 (DNA having the sequence), and encodes a proteinof a mammalian vascular endothelial cell, said protein having a functionof binding a modified low-density lipoprotein (namely a receptorthereof).

Moreover, the present invention relates to an antibody of a receptor formodified low-density lipoprotein corresponding to a DNA sequence setforth in SEQ ID NO: 1, 3 or 5 or an analogue thereof (or a receptor formodified low-density lipoprotein containing an amino acid sequence setforth in SEQ ID NOS: 2, 4 or 6 or an analogue thereof).

The DNA sequence of the present invention can be integrated into anexpression vector. Therefore, the present invention further provides aprocess for the production of mammalian vascular endothelial receptorfor modified lowensity lipoprotein or an analogue thereof, whichcomprises nserting the recombinant expression vector into a host celland culturing the cell under expression promoting conditions.

The invention furthermore provides a protein composition containing abiologically active mammalian vascular endothelial receptor for modifiedlow-density lipoprotein or an analogue thereof which is produced asmentioned above.

The obtained protein composition containing a biologically activemammalian vascular endothelial receptor for modified low-densitylipoprotein or an analogue thereof is effective in an assay of themammalian modified low-density lipoprotein. The composition can also beused in preparation of an antibody to the vascular endothelial receptorfor modified low-density lipoprotein. The antibody can be used indiagnosis.

It is apparent from the above-described biological activities of themodified low-density lipoprotein and the receptor thereof that an agentcontaining an antibody to the vascular endothelial receptor for modifiedlow-density lipoprotein is effective in diagnosis of atherosclerosis.

In the present specification, the term "receptor for modifiedlow-density lipoprotein" means proteins which are capable of bindingmodified low-density lipoprotein molecules and, in their nativeconfiguration as mammalian plasma membrane proteins, presumably play arole in transducing the signal provided by a modified low-densitylipoprotein to a vascular endothelial cell. In the specification, theterm includes analogues of native proteins with an activity of binding amodified low-density lipoprotein or a signal transducing activity.

The term "subtype of a receptor for modified low-density lipoprotein"means molecules of a receptor for modified low-density lipoprotein whichshow different pharmacological potency rank orders, namely differentaffinities or selectivities for various isopeptides of modifiedlow-density lipoprotein, such as oxidized low-density lipoprotein oracetylated low-density lipoprotein.

The term "essentially" used in the expression "a DNA sequenceessentially encoding a receptor for modified low-density lipoprotein" orthe like means that a particular subject sequence, for example, a mutantsequence, varies from a reference sequence by one or more substitutions,deletions, or additions, the net effect of which does not result in anadverse functional dissimilarity between subject sequence and referencesequence set forth in the SEQ ID NO: 1, 3 or 5.

In more detail, the sequence can be modified so long as a proteincorresponding to the sequence has a biological activity (describedbelow), namely an activity of binding modified low-density lipoprotein.Therefore, the region encoding a portion of binding modified low-densitylipoprotein should be the same as the reference sequence set forth inthe SEQ ID NO: 1, 3 or 5 except for variation due to code degeneracy andsubstitution of an amino acid to an analogous amino acid. The otherregion merely requires at least 30% (preferably at least 50%, and morepreferably at least 80%) similarity in the sequence.

The above-mentioned substitution to an analogous amino acid means aminoacid substitution in a group where the natural amino acids areclassified into the following eight groups.

(1) Monoaminomonocarboxylic acid Gly, Ala, Val, Leu, Ile

(2) Oxyamino acid Ser, Thr

(3) Sulfur-containing amino acid Cys, Met

(4) Monoaminodicarboxylic acid Asp, Glu

(5) Diaminomonocarboxylic acid Lys, Arg

(6) Aromatic amino acid Phe, Tyr

(7) Heterocyclic amino acid His, Trp, Pro

(8) Amide amino acid Asn, Gln

For purposes of determining similarity, truncation or internal deletionsof the reference sequence should be disregarded. Sequences having lesserdegrees of similarity, comparable biological activity, and equivalentexpression characteristics are considered to be essential equivalents.

The term "biologically active" used as a characteristic of a receptorfor modified low-density lipoprotein means either that a particularmolecule has sufficient amino acid sequence similarity with theembodiments of the present invention having an activity of bindingmodified low-density lipoprotein, or that a particular molecule hassufficient amino acid sequences similarity to the receptor for modifiedlow-density lipoprotein to be capable of transmitting stimulus of themodified low-density lipoprotein to cell as a component of hybridreceptor constructs.

In more detail, the affinity (dissociation constant) of a particularmolecule for standard oxidized low-density lipoprotein is not more than1 μM. In the present invention, the affinity preferably is not more than0.1 μM, and more preferably is not more than 0.01 μM.

The term "biologically active" also means that a particular molecule hasa function of accelerating internalization of modified low-densitylipoprotein into vascular endothelial cells.

The term "DNA sequence" means a DNA polymer, in the form of a separatefragment or as a component of larger DNA constructs. The DNA constructsare derived from DNA isolated at least once in essentially pure form(free of contaminating endogenous materials) and in a quantity orconcentration enabling identification, manipulation, and recovery of thesequence and its component nucleotides sequences by standard biochemicalmethods, for example, using a cloning vector. The DNA sequences arepreferably provided in the form of an open reading frame uninterruptedby internal nontranslated sequences, or introns, which are typicallypresent in eukaryotic genes. However, genomic DNA containing therelevant sequences can also be used. Sequences of non-translated DNA maybe present 5' or 3' from the open reading frame. The non-translated DNAdoes not interfere with manipulation or expression of the codingregions.

The term "recombinant expression vector" means a plasmid comprising atranscriptional unit. The unit comprises (a) a genetic element orelements having a regulatory role in gene expression, for example,promoters or enhancers, (b) a structural or coding sequence which istranscribed into MRNA and translated into protein, and (c) appropriatetranscription and translation initiation and termination sequences.Structural elements used in yeast expression systems preferably includea leader sequence enabling extracellular secretion of translated proteinby a host cell. In the case that a recombinant protein is expressedwithout a leader or transport sequence, it may include an N-terminalmethionine residue. This residue may optionally be cleaved from theexpressed recombinant protein to provide a final product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates a high expression lasmid vector, pME18Sused in Example 1.

FIG. 2 is a graph showing the fluorescent intensity distribution ofCOS-7 cells which have been transfected with pBLOX-1 and a control COS-7cell which has not been transfected. The intensity was measured by FACS.

FIG. 3 is a fluorescent micrograph showing increase of fluorescentintensity in cytoplasm caused by incubation with DiI-labeled modifiedlow-density lipoprotein.

PREFERRED EMBODIMENTS OF THE INVENTION

Next, isolation of cDNA encoding the receptor for modified low-densitylipoprotein and determination of the DNA sequence are described below.

A cDNA library was prepared by a reverse transcription of poly(A)⁺ RNA,which was isolated from cultured bovine aortic endothelial cells. A DNAsequence encoding a bovine receptor for modified low-density lipoproteinwas isolated from the cDNA library. The library was screened by directexpression of MRNA from DNA fragments accumulated in monkey COS-7 cellsusing a mammalian expression vector (pME18S). The vector containsregulatory sequences derived from SV40, human T lymphocyte leukemicvirus type I. Transfected COS-7 cells were incubated in a culture mediumcontaining DiI (1,1'-di-octadecyl-3,3,3',3'-tetramethylindocarbocyanineperchlorate)-labeled oxidized low-density lipoproteins. The cells werewashed to remove free DiI-labeled oxidized low-density lipoproteins. Thecells were subjected to tripsinization to suspend the cells. The cellswere treated in FACS (fluorescence-activated cell sorter) to measurefluorescence of DiI and to recover cells showing high fluorescentintensity. Plasmid was extracted from the transfected cells, and E. coliwas transformed with the plasmids. Plasmids were purified and theabove-mentioned procedures were repeated. The procedures were repeatedfour times to synthesize monoclonal surface protein having an activityof binding modified low-density lipoprotein. The clone was isolated, andthe insertion fragment sequence as examined to determine cDNA sequenceof a bovine receptor for modified low-density lipoprotein.

COS-7 cells were transfected with isolated cDNA clone to express thegene. As a result, the cells obtained a specific binding activity tooxidized low-density lipoprotein. The modified low-density lipoproteinis internalized into the cells. Further, the cells do not have anactivity of binding native (not modified) low-density lipoprotein.Accordingly, the receptor is considered specific to modified low-densitylipoprotein.

The above-determined DNA sequence encoding the receptor for modifiedlow-density lipoprotein are set forth in Sequence Nos. 1 and 3. Thecorresponding sequences are set forth in SEQ ID NOS: 2 and 4.

The DNA sequence and the amino acid sequence are described below.

As is shown in the SEQ ID NOS: 2 and 4, the receptor for modifiedlow-density lipoprotein has at least two sub-types. The SEQ ID NO: 4 isthe same as the SEQ ID NO: 2, except that three amino acids (Thr ThrGly) are inserted after the 24th amino acid (Gly) of the SEQ ID NO: 2.In the present specification, the sequence is described referring to theSEQ ID NO: 2 unless otherwise specified.

There is an open reading frame of 810bp encoding 270 amino acid residuesfrom the first ATG (initiation codon encoding methionine) to the stopcodon of TGA (811-813). The 3' nontranslated region in the mRNA of thereceptor for modified low-density lipoprotein encoded by this cDNAcontains seven AUUUA sequences which unstabilize mRNA. This is analogousto transiently expressed cytokine or growth factor.

The encoded polypeptide contains a stretch of 26 hydrophobic amino acidresidues (amino acid Nos. 31-56 in the SEQ ID NO: 2 and amino acid Nos.34-59 in the SEQ ID NO: 4), which are likely to represent atransmembrane domain. The C-terminal region after the putativetransmembrane domain contains four potential glycosylation sites (aminoacid Nos. 69, 135, 179 and 208 in the SEQ ID NO: 1 and amino acid Nos.72, 138, 182 and 211 in the SEQ ID NO: 4).

Further, a cDNA library was prepared by a reverse transcription ofpoly(A)⁺ RNA, which was extracted from human lung. A DNA sequenceencoding the human receptor for modified low-density lipoprotein wasisolated from the cDNA library. The library was screened according to aplaque hybridization method using XhoI/PstI fragments of pBMLR1, whichwas labeled with α-32P!dCTP. The hybridization was conducted at 55° C.in 50 mM Tris-HCl (pH 7.5), 1M NaCl, 1% SDS, 0.2 g/l Yeast tRNA. Afterthe hybrid was washed three times with 2xSSC/0.1% SDS for 15 minutes,positive clone was identified by an autoradiography. The clone wasisolated, and the insertion fragment sequence was examined to determinecDNA sequence of a human receptor for modified low-density lipoprotein.The sequence seemed a part of a domain encoding the protein. Therefore,cDNA encoding the whole protein was obtained from cDNA library preparedby a reverse transcription of poly(A)⁺ RNA, which was extracted fromhuman placenta. The procedures were conducted according to 5'-RACE(rapid amplification of cDNA end) method by using the partial sequence.

The SEQ ID NO: 5 shows the DNA sequence of the human receptor formodified low-density lipoprotein SEQ ID NO: 6 shows the amino acidsequence of the human receptor for modified low-density lipoprotein.

The human sequence as well as the bovine sequence has an open readingframe of 810 bp encoding 270 amino acid residues from the first ATG(initiation codon encoding methionine) to the stop codon of TGA(811-813).

The polypeptide encoded by the cDNA contains a stretch of 27 hydrophobicamino acid residues in analogy with bovine sequence, which are likely torepresent a transmembrane domain. The C-terminal region after theputative transmembrane domain contains four potential glycosylationsites (amino acid Nos. 69, 135, 179 and 206).

Each of the bovine and human amino acid sequences has the structure ofC-type lectin in an extracellular domain. The domain (amino acid Nos.140-270) is considered to have an activity of binding modifiedlow-density lipoprotein. Therefore, the peptide having the partialsequence of the amino acid Nos. 140-270 is also considered to have anactivity of binding modified low-density lipoprotein.

The present invention provides the above-described DNA sequence encodingthe receptor for modified low-density lipoprotein and the DNA sequenceencoding the partial sequence of the amino acid Nos. 140-270. The DNAsequence is preferably provided in a form which is capable of beingexpressed in a recombinant transcriptional unit under the control ofmammalian, microbial, viral transcriptional or translational controlelements. For example, a sequence to be expressed in a microorganismwill contain no introns. In a preferred embodiment, the DNA sequencecomprises at least one, but optionally more than one sequence componentderived from a cDNA sequence or copy thereof.

The sequences may be linked or flanked by DNA sequence prepared byassembly of synthetic oligonucleotides. However, synthetic genesassembled exclusively from oligonucleotides could be constructed usingthe sequence information provided herein. A representative sequencecontains those essentially identical to the nucleotide sequences setforth in the SEQ ID NOS: 1, 3 to 3. The coding sequences may includecodons encoding one or more additional amino acids located at theN-terminus, for example, an N-terminal ATG codons specifying methioninelinked with reading frame in the nucleotide sequence. Due to codedegeneracy, there can be considerable variation in nucleotide sequencesencoding the same amino acid sequence. Other embodiments includesequences capable of hybridizing to the representative sequence undermoderately stringent conditions (420° C., 20% (v/v) formamide). Theother sequences degenerate to those described above which encodebiologically active polypeptide of a receptor for modified low-densitylipoprotein.

The sequence can be expressed in a recombinant transcription unitcontaining an inducible regulatory element derived from an operon ofmicroorganism or virus. The present invention also provides expressionvectors for producing useful quantities of a purified receptor formodified low-density lipoprotein. The vectors can comprise synthetic orcDNA derived DNA fragments encoding a mammalian receptor for modifiedlow-density lipoprotein or bioequivalent homologues operably linked toregulatory elements derived from mammalian, bacterial, yeast,bacteriophage or viral genes. Useful regulatory elements are describedin greater detail below. Following transformation, transfection orinfection of appropriate cell lines, such vectors can be induced toexpress recombinant protein.

A mammalian receptor for modified low-density lipoprotein can beexpressed in mammalian cells, yeast, bacteria, or other cells under thecontrol of appropriate promoters. Cell-free translation systems couldalso be employed to produce the mammalian receptor for modifiedlow-density lipoprotein using mRNAs derived from the DNA constructs ofthe present invention. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts aredescribed by Pouwel et al. (Cloning Vectors: A Laboratory Manual,Elsevier, New York, 1985), the relevant disclosure of which is herebyincorporated by reference.

Various mammalian cell culture systems can be employed to expressrecombinant protein. Examples of suitable mammalian host cell linesinclude the COS-7 lines of monkey kidney cells, described by Gluzman(Cell 23:175, 1981), and other cell lines capable of expressing anappropriate vector, for example, C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors may comprise nontranscribed elementssuch as an origin of replication, a suitable promoter and enhancer, andother 5' or 3' flanking nontranscribed sequences, and 5' or 3'nontranslated sequences, such as necessary ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, and terminationsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 replication origin, early promoter, enhancer, splice, andpolyadenylation sites, may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. Exemplaryvectors can be constructed as disclosed by Okayama and Berg (Mol CellBiol. 3, 280, 1983).

A useful system for stable high level expression of mammalian receptorcDNAs in C127 rat mammary epithelial cells can be constructedessentially as described by Cosman et al. (Molecular Immunol. 23:935,1986).

Yeast systems, preferably employing Saccharomyces species such as S.cerevisiae, can also be employed for expression of the recombinantproteins of the present invention. Yeast of other genera, for example,Pichia or Kluyveromyces, has also been employed as production strainsfor recombinant proteins.

Generally, useful yeast vectors will include origins of replication andselectable markers permitting transformation of both yeast and E. coli,e.g., the ampicillin resistance gene (Amp^(r)) of E. coli and S.cerevisiae TRP1 gene, and a promoter derived from a highly expressedyeast gene to induce transcription of a downstream structural gene. Suchpromoters can be derived from yeast transcriptional units encodinghighly expressed genes such as 3-phosphoglycerate kinase (PGK),α-factor, acid phosphatase, or heat shock proteins, among others. Theheterologous structural sequence is assembled in appropriate readingframe with translation initiation and termination sequences, and,preferably, a leader sequence capable of directing secretion oftranslated protein into the extracellular medium. Optionally, theheterologous sequences can encode a fusion protein including anN-terminal identification peptide or other sequence imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product.

Useful yeast vectors can be assembled using DNA sequences from pBR322(Amp^(r) gene and origin of replication) for selection and replicationin E. coli and yeast DNA sequences including a glucose-repressiblealcohol dehydrogenase 2 (ADH2) promoter. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). Such vectors may also include a yeast TRP1gene as a selectable marker and the yeast 2μ origin of replication. Ayeast leader sequence, for example, the α-factor leader which directssecretion of heterologous proteins from a yeast host, can be insertedbetween the promoter and the structural gene to be expressed (see U.S.Pat. No. 4,546,082; Kurian et al., Cell 30:933, 1982); and Bittner etal., Proc. Natl. Acad. Sci. USA 81:983, 1984).

The leader sequence may be modified to contain, near its 3' end, one ormore useful restriction sites to facilitate fusion of the leadersequence to foreign genes.

Suitable yeast transformation protocols are known to those skilled inthe art; an exemplary technique is described by Hinnen et al. (Proc.Nati. Acad. Sci. USA 75:1929, 1978), selecting for Trp⁺ transformants ina selective medium consisting of 0.67% yeast nitrogen source, 0.5%casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil.

Host strains transformed by vectors comprising the ADH2 promoter may begrown for expression in a rich medium consisting of 1% yeast extract, 2%peptone and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Deregulation of the ADH2 promoter occurs upon exhaustion ofmedium glucose. Crude yeast supernatants are harvested by filtration andheld at 4° C. prior to further purification.

Useful expression vectors for bacterial use are constructed by insertinga DNA sequence encoding a mammalian receptor for modified low-densitylipoprotein together with suitable translation initiation andtermination signals in operable reading frame with a functionalpromoter. The vector will comprise one or more phenotypic selectablemarkers and an origin of replication to ensure growth within the host.Suitable prokaryotic hosts for transformation include E. coli, Bacillussubtilis, Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus, although others may alsobe employed as a matter of choice.

Expression vectors are conveniently constructed by cleavage of cDNAclones at sites close to the codon encoding the N-terminal residue ofthe mature protein. Synthetic oligonucleotides can then be used to "addback" any deleted sections of the coding region and to provide a linkingsequence for ligation of the coding fragment in appropriate readingframe in the expression vector, and optionally a codon specifying aninitiator methionine.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example pKK223-3p (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Projema Biotec, Madison, Wis,USA). These pBR322 "main chain" sections are combined with anappropriate promoter and the structural sequence to be expressed.

A particularly useful bacterial expression system employs the phageλP_(L) promoter and c1857 thermolabile repressor. Plasmid vectorsavailable from the American Type Culture Collection which incorporatederivatives of the λP_(L) promoter include plasmid pHUB2, resident in E.coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC53082). Other useful promoters for expression in E. coli include the T7RNA polymerase promoter described by Studier et al. (J. Mol. Biol.189:113, 1986), the lacZ promoter described by Lauer (J. Mol. Biol.Appl. Genet. 1:139-147, 1981) which is available as ATCC 37121, and thetac promoter described by Maniatis (Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, 1982, p412), which is availableas ATCC 37138.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification. Cells are grown, for example, in a 10 liter fermenteremploying conditions of maximum aeration and vigorous agitation. Anantifoaming agent (Antifoam A) is preferably employed. Cultures aregrown at 30° C. in the superinduction medium disclosed by Mott et al.(Proc. Natl. Acad. Sci. USA 82:88, 1985), alternatively includingantibiotics, derepressed at a cell density corresponding to A₆₀₀=0.4-0.5 by elevating the temperature to 42° C., and harvested for 2-20hours, preferably 3-6 hours after the upward temperature shift. The cellmass is initially concentrated by filtration or other means, thencentrifuged at 10,000×g (10,000 G) for 10 minutes at 4° C., followed byrapidly freezing the cell pellet.

Preferably, purified mammalian receptors for modified low-densitylipoprotein or bioequivalent analogues are prepared by culturingsuitable host/vector systems to express the recombinant translationproducts of the synthetic genes of the present invention, which are thenpurified from culture media.

An alternative process for producing a purified receptor for modifiedlow-density lipoprotein involves purification from cell culturesupernatants or extracts. In this approach, a cell line which elaboratesuseful quantities of the protein is employed. Supernatants from suchcell lines can be optionally concentrated using a commercially availableprotein concentration filter, for example, an Amicon or Millipore Falconultrafiltration unit. Following the concentration step, the concentratecan be applied to a suitable purification matrix as previouslydescribed. For example, a suitable affinity matrix can comprise areceptor for modified low-density lipoprotein or lectin or antibodymolecule bound to a suitable support. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups.

Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having methyl or other aliphatic groups, can beemployed to further purify a receptor composition for modifiedlow-density lipoprotein. Some or all of the foregoing purificationsteps, in various combinations, can also be employed to provide ahomogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or gel filtrationchromatography steps. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of recombinant mammalian receptor for modifiedlow-density lipoprotein can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption or useof cell lysing agents.

Fermentation of yeast which expresses a mammalian receptor for modifiedlow-density lipoprotein as a secreted protein greatly simplifiespurification. Secreted recombinant protein resulting from a large-scalefermentation can be purified by methods analogous to those disclosed byUrdal et al. (J. Chromatog. 296:171, 1984). This reference describes twosequential, reversed-phase HPLC steps for purification of recombinanthuman GM-CSF on a preparative HPLC column.

In its various embodiments, the present invention provides essentiallyhomogeneous polypeptides of a recombinant mammalian receptor formodified low-density lipoprotein free of contaminating endogenousmaterial.

Recombinant proteins of a receptor for modified low-density lipoproteinaccording to the present invention also include suitable peptide orprotein sequences employed as aids to expression in microorganisms orpurification of microbially expressed proteins.

Bioequivalent analogues of the proteins of this invention includevarious analogs, for example, truncated versions of a receptor formodified low-density lipoprotein wherein terminal residues or sequences,which exist in internal cell and are not needed for biological activity,are deleted.

As used herein, "mutant amino acid sequence" refers to a polypeptideencoded by a nucleotide sequence intentionally made variant from anative sequence. "Mutant protein" or "analog" means a protein comprisinga mutant amino acid sequence. "Native sequence" refers to an amino acidor nucleic acid sequence which is identical to a wild-type or nativeform of a gene or protein.

The protein of a receptor for modified low-density lipoprotein can bedigested with a protease to obtain a soluble peptide fragment. Thesoluble peptide fragment can also be obtained by expressing a part of areceptor for the modified low-density lipoprotein in E. coli or mammalsaccording to a recombinant DNA method. The obtained fragments are alsoincluded in the present invention so long as the fragments have thedefinition, namely an activity of binding a modified low-densitylipoprotein.

Using the obtained soluble receptor, a modified low-density lipoproteincan be inactivated by binding the lipoprotein with the soluble peptidefragment. Accordingly, the peptide fragment can be used to cure adisease caused by a modified low-density lipoprotein.

Examples of the present invention are described below. In the followingExamples, the amino acid sequence of the receptor for modifiedlow-density lipoprotein and the DNA sequence encoding the receptor wereelucidated from a bovine aortic endothelial cell. After the sequencesset forth in the SEQ ID NOS: 1 and 3 were elucidated, the amino acidsequence of a human endothelial receptor for modified low-densitylipoprotein and the DNA sequence encoding the receptor (SEQ ID NO: 5)were elucidated more easily. The amino acid sequence of the receptor formodified low-density lipoprotein and the DNA sequence encoding thereceptor can easily be elucidated from another mammalian endothelialcell in a similar manner.

In more detail, a DNA sequence encoding another mammalian endothelialreceptor for modified low-density lipoprotein can be selected frompoly(A)⁺ RNA, which was extracted from another mammalian endothelialcell or other organisms. The selection can be conducted by ahybridization with the DNA sequences set forth in the SEQ ID NOS: 1,3and 5. One skilled in the art can easily elucidate the sequence, as ismentioned above. The situation is now different from the Example 1 wherethere were no clues to the target sequence.

The obtained sequence can easily be analyzed by referring to the SEQ IDNOS 1, 3and 5. The analysis can be conducted much easier than theExample 1 (where there were no sequences to be referred).

In the following Examples, an assay was conducted with respect to anactivity of binding an oxidized low-density protein. The same assay canalso be conducted with respect to an activity of binding an acetylatedlow-density protein.

EXAMPLE 1

A cDNA library was constructed by a reverse transcription of mRNAincluding poly(A)⁺ RNA according to a procedure similar to that ofChomczynski et al. (Biotechniques 15, 532, 1993). The poly(A)⁺ RNA wasisolated from total RNA extracted from cultured bovine aorticendothelial cell. In more detail, the cells were dissolved in a solutionof acidic guanidinium isocyanate/phenol. Chloroform was added to thesolution, and the solution was centrifuged to separate an aqueous phaseand an organic phase. The aqueous phase was recovered, and purified withalcohol sedimentation. Poly(A⁺)RNA was isolated by oligo dT cellulosechromatography and double-stranded cDNA was prepared by a method similarto that of Gubler and Hoffman (Gene 25, 263, 1983). Briefly, the RNA wascopied into cDNA by reverse transcriptase using either oligo dT orrandom oligonucleotides as primer. The cDNA was made double-stranded byincubation with E. coli DNA polymerase I and RNase H, and the ends madeflush by further incubation with T4 DNA polymerase. BstXI linker wasadded to the blunt-ended cDNA, and then short chains were removed by agel filtration chromatography using Sephacryl S-500HR. The cDNA wassubcloned into a high expression plasmid vector for mammalian cells(pME18S). A schematic illustration of pME18S is shown in FIG. 1(obtained from Dr. Maruyama of Tokyo Medical and Dental University).

The pME18S vector is a plasmid vector of 3.4 kb which contains areplication initiating point of SV 40 and a type I promoter ofSV40/human T lymphocyte leukemic virus.

The bovine aortic cDNA library on pME18S was used to transform E. coli(ElectroMax DH 10B) to provide about 7×10⁵ colonies. These recombinantswere cultured in 500 ml of 2xYT at 37° C. The plasmid DNA was preparedby a CsCl density-gradient centrifugation. The prepared DNA wastransfected into a sub-confluent mono-layer of monkey COS-7 cells usingLipofectamine. The cells were then grown in culture for three days topermit transient expression of the inserted sequences. The cellmonolayers in a plate were assayed for modified low-density lipoproteinuptake as follows.

To the plate was added 5 ml of DMEM medium with 10% fetal bovine serum(FBS) containing 15 μg of oxidized low-density lipoprotein labeled withDiI, and the plate was incubated for 12 hours at 37° C. at 5% CO₂. Thismedium was then discarded, and the plate was twice washed with PBS (pH7.4). The cells were separated from the plate by tripsinization. Thecells were applied to FACS to measure fluorescence of DiI and to recoverthe cells showing high fluorescent intensity. Plasmid was extracted fromthe recovered cells. The procedures were repeated four times. About7×10⁵ recombinants were screened from the library as is mentioned above.Thus COS-7 cells were transfected with a single clone pBLOX-1 which iscapable of inducing expression of a receptor for oxidized low-densitylipoprotein.

FIG. 2 is a graph showing the fluorescent intensity distribution ofCOS-7 cells which have been transfected with pBLOX-1 and a control COS-7cell which has not been transfected. The intensity was measured by FACS.

The inserted fragments of clone BLOX were subcloned to BluescriptIISK-plasmid, and its DNA sequence was determined according to the dideoxymethod (see Sanger et al., Proc. Natl. Acad. Sci. USA, 74, 5463, 1977).

EXAMPLE 2

Expression in tissues by northern blots using receptor cDNA

Poly(A)⁺ RNA was extracted from various bovine tissues in the samemanner as is mentioned above. Each 5 μg of the RNAs was separated usingformaldehyde/1.1% agarose gel electrophoresis, and transferred to genescreen plus membrane (NEN, DuPont). Then, 1.8 kb of cDNA fragments waslabeled by α-³² P-dCTP according to a random priming method to 8×10⁸c.p.m./mg, and used as a probe. Hybridization was carried out at 60° C.in a solution of 1M sodium chloride, 1% SDS and 250 μg salmon sperm DNA.The membrane was washed with 2× SSC/1% SDS. Autoradiography was carriedout for 8 hours.

Northern blots were carried out with respect to poly(A)⁺ RNAs extractedfrom 11 bovine tissues. As the results, a large amount of modifiedlow-density lipoprotein receptor mRNA was expressed in culturedendothelial cells and lung.

EXAMPLE 3

Expression of a receptor for modified low-density lipoprotein by CHO-K1cell

The cells expressing a receptor for modified low-density lipoproteinwere prepared using an expression vector pSV2bsr and the modifiedlow-density lipoprotein receptor expression plasmid pBLOX-1. The pSV2bsrvector contains a bs^(r) (blasticidin S-resistance) gene and a promoterderived from SV40 virus.

CHO (chinese hamster ovary) K1 cells were cultured as a subconfluentmonolayer in a HamF12 medium containing 10% FBS.

The CHO-K1 cells were transfected with the modified low-densitylipoprotein receptor expression plasmid pBLOX-1 and pSV2bsr usingLipofectamine. After 24 hours, the transfected cells were subcultured inan area about 10 times as large as the previous area. After 24 hours,the cells were well adhered, and the medium was replaced with a mediumcontaining 5 μg of blasticidin S.

Thus only the cells transfected with bsr gene into genome grew, andtheir colony was formed.

The well grown colonies were detached using trypsin according to thepenicillin cup method, and subcultured in 12 well plate using the sameDMEM medium.

Thus isolated clonal cells expressed a receptor for modified low-densitylipoprotein, which was confirmed by a fluorescent microscope. Theincrease of fluorescent intensity was observed by the microscope. Theincrease was caused by incorporation of modified low-density lipoproteinlabeled with DiI. FIG. 3 is the fluorescent micrograph.

EXAMPLE 4

Preparation of soluble modified low-density lipoprotein receptor

A cDNA fragment covering the extracellular domain of modifiedlow-density lipoprotein (Base Nos. 160-813 in the SEQ ID NO: 1) wasamplified by PCR with a pair of primers(5'-gcggatcctgtgctctcaatagattcgc-3' and5'-ggggatcctgatctcataaagaaacag-3' SEQ ID NO: 7 and SEQ ID NO: 8,respectively) tagged with a BamHI restriction site. Amplified fragmentwas digested with BamHI and subcloned into the BamHI site of pQE10(Qiagen), which expresses a protein tagged with six repeats of histidinein E. coli. The plasmid was transformed into an E. coli strain,XL-2Blue, and was cultured while shaking at 37° C. in 2xYT medium. Whenthe absorbance at 600 nm was 0.6, 1 mM of IPTG(isopropylthio-β-D-galactoside) was added to the medium. The culture wasfurther continued at 30° C. for 20 hours. E. coli was recovered by acentrifugation, was dissolved in 6M guanidine hydrochloride, 0.1M sodiumdihydrogen phosphate and 0.01M Tris (pH 8.0). Insoluble materials wereremoved by a centrifugation, and soluble modified low-densitylipoprotein receptor was adsorbed on Ni-NTA Agarose (Qiagen). Ni-NTAAgarose was washed with 8M urea, 0.1M sodium dihydrogen phosphate and0.01M Tris (pH 8.0) and with 8M urea, 0.1M sodium dihydrogen phosphateand 0.01M Tris (pH 6.3). Soluble modified low-density lipoproteinreceptor was eluted and purified with SM urea, 0.1M sodium dihydrogenphosphate, 0.01M Tris and 0.1M EDTA (pH 6.3). The soluble modifiedlow-density lipoprotein receptor was confirmed to be a homogeneoussample by using SDS-PAGE.

The other tags such as GST or c-myc can be used. The other purificationmethods such as a method using an antibody can also be used. Further,soluble modified low-density lipoprotein receptor can be prepared byrecombinant DNA procedures using an appropriate mammalian expressionvector.

EXAMPLE 5

Preparation of anti-LOX-1 antibody

The sequence encoding the extracellular domain (amino acids 61-270) ofbovine LOX-1 cDNA was amplified by a polymerase chain reaction with apair of primers (5'-ggggatcctgatctcataaagaaacag-3' and5'-gcggatcctgtgctctcaatagattcgc-3'SEQ ID NO: 7 and SEQ ID NO: 8,respectively) tagged with a BamHI restriction site. The amplified cDNAfragment was digested with BamHI, and subcloned into BamHI sites ofpQE10 vector (Qiagen). Protein synthesis and purification of theextracellular domain were conducted in QIA express system (Qiagen). Inmore detail, an E. coli strain, XL-2 blue (Stratagene) was transformedby the plasmid, and was cultured in 2xYT medium. The protein synthesiswas induced with isopropylthio-β-D-galactoside. The cells were recoveredby a centrifugation, and dissolved in 6M guanidine hydrochloride, 0.1Msodium phosphate and 0.01M Tris HCl (pH 8.0). A column chromatographywas conducted with Ni-NTA resin column (Qiagen). The column was washedwith 8M urea, 0.1M sodium phosphate and 0.01M Tris HCl (pH 6.3). Proteinwas eluted with 8M urea, 0.1M EDTA, 0.1M sodium phosphate and 0.01M TrisHCl (pH 6.3). The buffer was replaced with saline buffered with aphosphate salt by Centriprep 10 (Amicon). The protein was emulsifiedwith the same volume of complete Freund's adjuvant. Rabbits wereimmunized by intracutaneous injection of the emulsion into the skinbetween blade bone and spine every two weeks.

Immunoblot

Cultured bovine aortic endothelial cells were directly solubilized in asample buffer of SDS-PAGE. The extracts were separated by SDS-PAGE andblotted onto nylon membranes. After blocking with Block Ace (Snow BrandMilk Products Co., Ltd.), immunostaining of the membranes with anantibody, which was obtained from the above-mentioned rabbits, wasperformed using peroxidase-conjugated avidinbiotin complex andimmunostain kit (Vector).

INDUSTRIAL APPLICABILITY

An object of the present invention is to elucidate the structure of avascular endothelial receptor for modified low-density lipoprotein andto thereby provide a DNA sequence encoding the vascular endothelialreceptor for modified low-density lipoprotein.

Another object of the invention is to provide a process for productionof a vascular endothelial receptor for modified low-density lipoproteinor an analogue thereof.

A further object of the invention is to provide a protein compositioncontaining vascular endothelial receptor for modified low-densitylipoprotein or an analogue thereof.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 8    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1897 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Bos tauru - #s              (F) TISSUE TYPE: Vascul - #ar endothelial cells    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: Bovine aor - #tic endothelial cell cDNA              (B) CLONE: pBLOX-1    -     (ix) FEATURE:              (A) NAME/KEY: polyA.sub.-- - #site              (B) LOCATION: 1880..1897    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #RNA              (B) LOCATION: 1859..1864    #/function= "PolyA Signal"ATION:    -     (ix) FEATURE:              (A) NAME/KEY: 5'UTR              (B) LOCATION: 1..34    -     (ix) FEATURE:              (A) NAME/KEY: 3'UTR              (B) LOCATION: 848..1897    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 35..847    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - GCTTCACTCT CTCATTCTTG GAATACATTT GAAA ATG ACT GTT G - #AT GAC CCC      52    #  Met Thr Val Asp Asp Pro    # 5  1    - AAG GGT ATG AAA GAT CAA CTT GAT CAG AAG CC - #A AAT GGC AAG ACA GCA     100    Lys Gly Met Lys Asp Gln Leu Asp Gln Lys Pr - #o Asn Gly Lys Thr Ala    #             20    - AAA GGT TTT GTT TCC TCT TGG AGG TGG TAC CC - #T GCT GCT GTG ACT CTA     148    Lys Gly Phe Val Ser Ser Trp Arg Trp Tyr Pr - #o Ala Ala Val Thr Leu    #         35    - GGG GTC CTT TGT CTG GGA TTA CTG GTG ACT GT - #T ATA TTG TTG ATA CTG     196    Gly Val Leu Cys Leu Gly Leu Leu Val Thr Va - #l Ile Leu Leu Ile Leu    #     50    - CAA TTA TCC CAG GTC TCT GAT CTC ATA AAG AA - #A CAG CAA GCA AAT ATT     244    Gln Leu Ser Gln Val Ser Asp Leu Ile Lys Ly - #s Gln Gln Ala Asn Ile    # 70    - ACT CAC CAG GAA GAT ATC CTG GAG GGA CAG AT - #T TTA GCC CAG CGC CGA     292    Thr His Gln Glu Asp Ile Leu Glu Gly Gln Il - #e Leu Ala Gln Arg Arg    #                 85    - TCA GAA AAA TCT GCC CAG GAG TCA CAG AAG GA - #A CTC AAA GAA ATG ATA     340    Ser Glu Lys Ser Ala Gln Glu Ser Gln Lys Gl - #u Leu Lys Glu Met Ile    #            100    - GAA ACC CTT GCC CAC AAG CTG GAT GAG AAA TC - #C AAG AAA CTA ATG GAA     388    Glu Thr Leu Ala His Lys Leu Asp Glu Lys Se - #r Lys Lys Leu Met Glu    #       115    - CTT CAC CGC CAG AAC CTG AAT CTC CAA GAA GT - #T CTG AAA GAG GCA GCA     436    Leu His Arg Gln Asn Leu Asn Leu Gln Glu Va - #l Leu Lys Glu Ala Ala    #   130    - AAC TAT TCA GGT CCT TGT CCC CAA GAC TGG CT - #C TGG CAT GAA GAA AAC     484    Asn Tyr Ser Gly Pro Cys Pro Gln Asp Trp Le - #u Trp His Glu Glu Asn    135                 1 - #40                 1 - #45                 1 -    #50    - TGT TAC CAA TTT TCC TCT GGC TCT TTT AAT TG - #G GAA AAA AGC CAG GAG     532    Cys Tyr Gln Phe Ser Ser Gly Ser Phe Asn Tr - #p Glu Lys Ser Gln Glu    #               165    - AAC TGC TTG TCT TTG GAT GCC CAC TTG CTG AA - #G ATT AAT AGC ACA GAT     580    Asn Cys Leu Ser Leu Asp Ala His Leu Leu Ly - #s Ile Asn Ser Thr Asp    #           180    - GAA CTG GAA TTC ATC CAG CAA ATG ATT GCC CA - #T TCC AGT TTC CCC TTC     628    Glu Leu Glu Phe Ile Gln Gln Met Ile Ala Hi - #s Ser Ser Phe Pro Phe    #       195    - TGG ATG GGG TTG TCA ATG AGG AAA CCC AAT TA - #C TCG TGG CTT TGG GAA     676    Trp Met Gly Leu Ser Met Arg Lys Pro Asn Ty - #r Ser Trp Leu Trp Glu    #   210    - GAT GGT ACT CCT TTG ACG CCC CAC TTG TTT AG - #A ATT CAG GGA GCT GTT     724    Asp Gly Thr Pro Leu Thr Pro His Leu Phe Ar - #g Ile Gln Gly Ala Val    215                 2 - #20                 2 - #25                 2 -    #30    - TCC CGT ATG TAT CCT TCA GGG ACC TGT GCA TA - #T ATT CAA AGG GGA ACT     772    Ser Arg Met Tyr Pro Ser Gly Thr Cys Ala Ty - #r Ile Gln Arg Gly Thr    #               245    - GTT TTT GCT GAA AAC TGC ATT TTA ACT GCA TT - #C AGT ATA TGT CAA AAG     820    Val Phe Ala Glu Asn Cys Ile Leu Thr Ala Ph - #e Ser Ile Cys Gln Lys    #           260    - AAG GCG AAT CTA TTG AGA GCA CAG TGA ATTTGAAGG - #A TCTGGAGGAA     867    Lys Ala Asn Leu Leu Arg Ala Gln  *    #       270    - AAGAAGGAAA CCTTTGAATT CTCTTCTGGA ATTTAAGCTA TACTTCATCA CT - #TAGATGTA     927    - AACCATTAGA GCCCAGGGAA ATGCCTGCTA CTGGTTGAGT GCAGAACTCC TT - #AGCAGAGA     987    - CTGGCCCAGC TGCCTGGCAC CTTGATAGCA AAAGTTGCAA TTCCCTCTGT AT - #ATTTTTCC    1047    - CTAACTTGTT CCAAGTCCTC CCCTGCAGGA CTTCAGAGAA GTCAATTTTT CT - #GTTTCCAT    1107    - TGTTTCTAAG AACTTGTTGC CTAACTCAAG GTCACAGCAT TTTTCTCACT TT - #TGTCCTAT    1167    - GCTTTCTTCT AGGCATTGTA GAGTTTTAGA TTTTACATGG AAATCTAGAA CT - #TATTTTAG    1227    - ATTAATTTCT AAGTGATATA TGGATGTATG GAAGTTTTCT GTTTGTTTTT TG - #CTTGTGAG    1287    - TATTCAATTG TTTTTGCAAC ATTTGCTGAA AAGACTATTC TTCCTTCACT AC - #ATTGCCTT    1347    - TGCACTGTTG TCAACAATTA TCCATACATG CCTGGCTCTA TTTCTGGATT TT - #CTATTCCT    1407    - TTCCATTTAT TTATTTATTA TTCTTGGCTT ACAACATCAC CATGATATTT TG - #AATTCTAT    1467    - GGTTCTTTAA TATATCTTGG AATCACATGG TAGTAGTTAT TCATTGTTGT TC - #TTTTTTAG    1527    - AGTTGTTTGG TTAATCTATG CTTTTGTATT TCTGTCTTAA ATTGGCTTGT CC - #ATTTCTAA    1587    - AAAAACTTGA AATTTTGAAT TGCACTGAAT CCATACATAA ATTTAGGGAA AA - #TTGAATTC    1647    - TTAAAAATAC TGATTTGTTC AACTCATGAA AAAGGTGTAT TGCTCTATTT AG - #GTATTCCT    1707    - TATTTTCTTT AAGCAATGCT TTTTAATGTT CTTTGTGTAG ATATTGTTAG AT - #TATCATCA    1767    - TGTATTTCAC ATTATTTATG CTACTGTAGA TAGTATTGTT ATCATTTGTT GT - #TCTTATTT    1827    - TCAAAGTCTT CTGCTAGTAT GTAGAATTAT AATAAAGTTT GATATTAATA TT - #AAAAAAAA    1887    #      1897    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  270 ami - #no acids              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Met Thr Val Asp Asp Pro Lys Gly Met Lys As - #p Gln Leu Asp Gln Lys    #                 15    - Pro Asn Gly Lys Thr Ala Lys Gly Phe Val Se - #r Ser Trp Arg Trp Tyr    #             30    - Pro Ala Ala Val Thr Leu Gly Val Leu Cys Le - #u Gly Leu Leu Val Thr    #         45    - Val Ile Leu Leu Ile Leu Gln Leu Ser Gln Va - #l Ser Asp Leu Ile Lys    #     60    - Lys Gln Gln Ala Asn Ile Thr His Gln Glu As - #p Ile Leu Glu Gly Gln    # 80    - Ile Leu Ala Gln Arg Arg Ser Glu Lys Ser Al - #a Gln Glu Ser Gln Lys    #                 95    - Glu Leu Lys Glu Met Ile Glu Thr Leu Ala Hi - #s Lys Leu Asp Glu Lys    #           110    - Ser Lys Lys Leu Met Glu Leu His Arg Gln As - #n Leu Asn Leu Gln Glu    #       125    - Val Leu Lys Glu Ala Ala Asn Tyr Ser Gly Pr - #o Cys Pro Gln Asp Trp    #   140    - Leu Trp His Glu Glu Asn Cys Tyr Gln Phe Se - #r Ser Gly Ser Phe Asn    145                 1 - #50                 1 - #55                 1 -    #60    - Trp Glu Lys Ser Gln Glu Asn Cys Leu Ser Le - #u Asp Ala His Leu Leu    #               175    - Lys Ile Asn Ser Thr Asp Glu Leu Glu Phe Il - #e Gln Gln Met Ile Ala    #           190    - His Ser Ser Phe Pro Phe Trp Met Gly Leu Se - #r Met Arg Lys Pro Asn    #       205    - Tyr Ser Trp Leu Trp Glu Asp Gly Thr Pro Le - #u Thr Pro His Leu Phe    #   220    - Arg Ile Gln Gly Ala Val Ser Arg Met Tyr Pr - #o Ser Gly Thr Cys Ala    225                 2 - #30                 2 - #35                 2 -    #40    - Tyr Ile Gln Arg Gly Thr Val Phe Ala Glu As - #n Cys Ile Leu Thr Ala    #               255    - Phe Ser Ile Cys Gln Lys Lys Ala Asn Leu Le - #u Arg Ala Gln    #           270    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1906 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Bos tauru - #s              (F) TISSUE TYPE: Vascul - #ar endothelial cells    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: Bovine aor - #tic edothelial cells cDNA              (B) CLONE: pBLOX-1    -     (ix) FEATURE:              (A) NAME/KEY: polyA.sub.-- - #site              (B) LOCATION: 1889..1906    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #RNA              (B) LOCATION: 1864..1873    #/function= "PolyA Signal"ATION:    -     (ix) FEATURE:              (A) NAME/KEY: 5'UTR              (B) LOCATION: 1..34    -     (ix) FEATURE:              (A) NAME/KEY: 3'UTR              (B) LOCATION: 857..1906    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 35..856    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - GCTTCACTCT CTCATTCTTG GAATACATTT GAAA ATG ACT GTT G - #AT GAC CCC      52    #  Met Thr Val Asp Asp Pro    # 5  1    - AAG GGT ATG AAA GAT CAA CTT GAT CAG AAG CC - #A AAT GGC AAG ACA GCA     100    Lys Gly Met Lys Asp Gln Leu Asp Gln Lys Pr - #o Asn Gly Lys Thr Ala    #             20    - AAA GGT ACT ACA GGT TTT GTT TCC TCT TGG AG - #G TGG TAC CCT GCT GCT     148    Lys Gly Thr Thr Gly Phe Val Ser Ser Trp Ar - #g Trp Tyr Pro Ala Ala    #         35    - GTG ACT CTA GGG GTC CTT TGT CTG GGA TTA CT - #G GTG ACT GTT ATA TTG     196    Val Thr Leu Gly Val Leu Cys Leu Gly Leu Le - #u Val Thr Val Ile Leu    #     50    - TTG ATA CTG CAA TTA TCC CAG GTC TCT GAT CT - #C ATA AAG AAA CAG CAA     244    Leu Ile Leu Gln Leu Ser Gln Val Ser Asp Le - #u Ile Lys Lys Gln Gln    # 70    - GCA AAT ATT ACT CAC CAG GAA GAT ATC CTG GA - #G GGA CAG ATT TTA GCC     292    Ala Asn Ile Thr His Gln Glu Asp Ile Leu Gl - #u Gly Gln Ile Leu Ala    #                 85    - CAG CGC CGA TCA GAA AAA TCT GCC CAG GAG TC - #A CAG AAG GAA CTC AAA     340    Gln Arg Arg Ser Glu Lys Ser Ala Gln Glu Se - #r Gln Lys Glu Leu Lys    #            100    - GAA ATG ATA GAA ACC CTT GCC CAC AAG CTG GA - #T GAG AAA TCC AAG AAA     388    Glu Met Ile Glu Thr Leu Ala His Lys Leu As - #p Glu Lys Ser Lys Lys    #       115    - CTA ATG GAA CTT CAC CGC CAG AAC CTG AAT CT - #C CAA GAA GTT CTG AAA     436    Leu Met Glu Leu His Arg Gln Asn Leu Asn Le - #u Gln Glu Val Leu Lys    #   130    - GAG GCA GCA AAC TAT TCA GGT CCT TGT CCC CA - #A GAC TGG CTC TGG CAT     484    Glu Ala Ala Asn Tyr Ser Gly Pro Cys Pro Gl - #n Asp Trp Leu Trp His    135                 1 - #40                 1 - #45                 1 -    #50    - GAA GAA AAC TGT TAC CAA TTT TCC TCT GGC TC - #T TTT AAT TGG GAA AAA     532    Glu Glu Asn Cys Tyr Gln Phe Ser Ser Gly Se - #r Phe Asn Trp Glu Lys    #               165    - AGC CAG GAG AAC TGC TTG TCT TTG GAT GCC CA - #C TTG CTG AAG ATT AAT     580    Ser Gln Glu Asn Cys Leu Ser Leu Asp Ala Hi - #s Leu Leu Lys Ile Asn    #           180    - AGC ACA GAT GAA CTG GAA TTC ATC CAG CAA AT - #G ATT GCC CAT TCC AGT     628    Ser Thr Asp Glu Leu Glu Phe Ile Gln Gln Me - #t Ile Ala His Ser Ser    #       195    - TTC CCC TTC TGG ATG GGG TTG TCA ATG AGG AA - #A CCC AAT TAC TCG TGG     676    Phe Pro Phe Trp Met Gly Leu Ser Met Arg Ly - #s Pro Asn Tyr Ser Trp    #   210    - CTT TGG GAA GAT GGT ACT CCT TTG ACG CCC CA - #C TTG TTT AGA ATT CAG     724    Leu Trp Glu Asp Gly Thr Pro Leu Thr Pro Hi - #s Leu Phe Arg Ile Gln    215                 2 - #20                 2 - #25                 2 -    #30    - GGA GCT GTT TCC CGT ATG TAT CCT TCA GGG AC - #C TGT GCA TAT ATT CAA     772    Gly Ala Val Ser Arg Met Tyr Pro Ser Gly Th - #r Cys Ala Tyr Ile Gln    #               245    - AGG GGA ACT GTT TTT GCT GAA AAC TGC ATT TT - #A ACT GCA TTC AGT ATA     820    Arg Gly Thr Val Phe Ala Glu Asn Cys Ile Le - #u Thr Ala Phe Ser Ile    #           260    - TGT CAA AAG AAG GCG AAT CTA TTG AGA GCA CA - #G TGA ATTTGAAGGA     866    Cys Gln Lys Lys Ala Asn Leu Leu Arg Ala Gl - #n  *    #       270    - TCTGGAGGAA AAGAAGGAAA CCTTTGAATT CTCTTCTGGA ATTTAAGCTA TA - #CTTCATCA     926    - CTTAGATGTA AACCATTAGA GCCCAGGGAA ATGCCTGCTA CTGGTTGAGT GC - #AGAACTCC     986    - TTAGCAGAGA CTGGCCCAGC TGCCTGGCAC CTTGATAGCA AAAGTTGCAA TT - #CCCTCTGT    1046    - ATATTTTTCC CTAACTTGTT CCAAGTCCTC CCCTGCAGGA CTTCAGAGAA GT - #CAATTTTT    1106    - CTGTTTCCAT TGTTTCTAAG AACTTGTTGC CTAACTCAAG GTCACAGCAT TT - #TTCTCACT    1166    - TTTGTCCTAT GCTTTCTTCT AGGCATTGTA GAGTTTTAGA TTTTACATGG AA - #ATCTAGAA    1226    - CTTATTTTAG ATTAATTTCT AAGTGATATA TGGATGTATG GAAGTTTTCT GT - #TTGTTTTT    1286    - TGCTTGTGAG TATTCAATTG TTTTTGCAAC ATTTGCTGAA AAGACTATTC TT - #CCTTCACT    1346    - ACATTGCCTT TGCACTGTTG TCAACAATTA TCCATACATG CCTGGCTCTA TT - #TCTGGATT    1406    - TTCTATTCCT TTCCATTTAT TTATTTATTA TTCTTGGCTT ACAACATCAC CA - #TGATATTT    1466    - TGAATTCTAT GGTTCTTTAA TATATCTTGG AATCACATGG TAGTAGTTAT TC - #ATTGTTGT    1526    - TCTTTTTTAG AGTTGTTTGG TTAATCTATG CTTTTGTATT TCTGTCTTAA AT - #TGGCTTGT    1586    - CCATTTCTAA AAAAACTTGA AATTTTGAAT TGCACTGAAT CCATACATAA AT - #TTAGGGAA    1646    - AATTGAATTC TTAAAAATAC TGATTTGTTC AACTCATGAA AAAGGTGTAT TG - #CTCTATTT    1706    - AGGTATTCCT TATTTTCTTT AAGCAATGCT TTTTAATGTT CTTTGTGTAG AT - #ATTGTTAG    1766    - ATTATCATCA TGTATTTCAC ATTATTTATG CTACTGTAGA TAGTATTGTT AT - #CATTTGTT    1826    - GTTCTTATTT TCAAAGTCTT CTGCTAGTAT GTAGAATTAT AATAAAGTTT GA - #TATTAATA    1886    #                 190 - #6    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  273 ami - #no acids              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - Met Thr Val Asp Asp Pro Lys Gly Met Lys As - #p Gln Leu Asp Gln Lys    #                 15    - Pro Asn Gly Lys Thr Ala Lys Gly Thr Thr Gl - #y Phe Val Ser Ser Trp    #             30    - Arg Trp Tyr Pro Ala Ala Val Thr Leu Gly Va - #l Leu Cys Leu Gly Leu    #         45    - Leu Val Thr Val Ile Leu Leu Ile Leu Gln Le - #u Ser Gln Val Ser Asp    #     60    - Leu Ile Lys Lys Gln Gln Ala Asn Ile Thr Hi - #s Gln Glu Asp Ile Leu    # 80    - Glu Gly Gln Ile Leu Ala Gln Arg Arg Ser Gl - #u Lys Ser Ala Gln Glu    #                 95    - Ser Gln Lys Glu Leu Lys Glu Met Ile Glu Th - #r Leu Ala His Lys Leu    #           110    - Asp Glu Lys Ser Lys Lys Leu Met Glu Leu Hi - #s Arg Gln Asn Leu Asn    #       125    - Leu Gln Glu Val Leu Lys Glu Ala Ala Asn Ty - #r Ser Gly Pro Cys Pro    #   140    - Gln Asp Trp Leu Trp His Glu Glu Asn Cys Ty - #r Gln Phe Ser Ser Gly    145                 1 - #50                 1 - #55                 1 -    #60    - Ser Phe Asn Trp Glu Lys Ser Gln Glu Asn Cy - #s Leu Ser Leu Asp Ala    #               175    - His Leu Leu Lys Ile Asn Ser Thr Asp Glu Le - #u Glu Phe Ile Gln Gln    #           190    - Met Ile Ala His Ser Ser Phe Pro Phe Trp Me - #t Gly Leu Ser Met Arg    #       205    - Lys Pro Asn Tyr Ser Trp Leu Trp Glu Asp Gl - #y Thr Pro Leu Thr Pro    #   220    - His Leu Phe Arg Ile Gln Gly Ala Val Ser Ar - #g Met Tyr Pro Ser Gly    225                 2 - #30                 2 - #35                 2 -    #40    - Thr Cys Ala Tyr Ile Gln Arg Gly Thr Val Ph - #e Ala Glu Asn Cys Ile    #               255    - Leu Thr Ala Phe Ser Ile Cys Gln Lys Lys Al - #a Asn Leu Leu Arg Ala    #           270    - Gln    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1318 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Homo Sapi - #ens    #placenta (F) TISSUE TYPE: Lung,    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: Human lung - # cDNA              (B) CLONE: lambdahLOX-1    -     (ix) FEATURE:              (A) NAME/KEY: 5'UTR              (B) LOCATION: 66..125    -     (ix) FEATURE:              (A) NAME/KEY: 3'UTR              (B) LOCATION: 949..1309    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 127..948    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - GGGGCCGCAC TAGTGATTCT GGTTCGGCCC ACCTCTGAAG GTTCCAGAAT CG - #ATAGTGAA      60    - TTCGTGATTT TAGTTTGTTG AAGTTCGTGA CTGCTTCACT CTCTCATTCT TA - #GCTTGAAT     120    #ACT GTG AAG GAC CAG       168TA AAG ATC CAG           Met Thr Phe Asp Asp Leu Ly - #s Ile Gln Thr Val Lys Asp Gln    #      10    - CCT GAT GAG AAG TCA AAT GGA AAA AAA GCT AA - #A GGT CTT CAG TTT CTT     216    Pro Asp Glu Lys Ser Asn Gly Lys Lys Ala Ly - #s Gly Leu Gln Phe Leu    # 30    - TAC TCT CCA TGG TGG TGC CTG GCT GCT GCG AC - #T CTA GGG GTC CTT TGC     264    Tyr Ser Pro Trp Trp Cys Leu Ala Ala Ala Th - #r Leu Gly Val Leu Cys    #                 45    - CTG GGA TTA GTA GTG ACC ATT ATG GTG CTG GG - #C ATG CAA TTA TCC CAG     312    Leu Gly Leu Val Val Thr Ile Met Val Leu Gl - #y Met Gln Leu Ser Gln    #             60    - GTG TCT GAC CTC CTA ACA CAA GAG CAA GCA AA - #C CTA ACT CAC CAG AAA     360    Val Ser Asp Leu Leu Thr Gln Glu Gln Ala As - #n Leu Thr His Gln Lys    #         75    - AAG AAA CTG GAG GGA CAG ATC TCA GCC CGG CA - #A CAA GCA GAA GAA GCT     408    Lys Lys Leu Glu Gly Gln Ile Ser Ala Arg Gl - #n Gln Ala Glu Glu Ala    #     90    - TCA CAG GAG TCA GAA AAC GAA CTC AAG GAA AT - #G ATA GAA ACC CTT GCT     456    Ser Gln Glu Ser Glu Asn Glu Leu Lys Glu Me - #t Ile Glu Thr Leu Ala    #110    - CGG AAG CTG AAT GAG AAA TCC AAA GAG CAA AT - #G GAA CTT CAC CAC CAG     504    Arg Lys Leu Asn Glu Lys Ser Lys Glu Gln Me - #t Glu Leu His His Gln    #               125    - AAT CTG AAT CTC CAA GAA ACA CTG AAG AGA GT - #A GCA AAT TGT TCA GCT     552    Asn Leu Asn Leu Gln Glu Thr Leu Lys Arg Va - #l Ala Asn Cys Ser Ala    #           140    - CCT TGT CCG CAA GAC TGG ATC TGG CAT GGA GA - #A AAC TGT TAC CTA TTT     600    Pro Cys Pro Gln Asp Trp Ile Trp His Gly Gl - #u Asn Cys Tyr Leu Phe    #       155    - TCC TCG GGC TCA TTT AAC TGG GAA AAG AGC CA - #A GAG AAG TGC TTG TCT     648    Ser Ser Gly Ser Phe Asn Trp Glu Lys Ser Gl - #n Glu Lys Cys Leu Ser    #   170    - TTG GAT GCC AAG TTG CTG AAA ATT AAT AGC AC - #A GCT GAT CTG GAC TTC     696    Leu Asp Ala Lys Leu Leu Lys Ile Asn Ser Th - #r Ala Asp Leu Asp Phe    175                 1 - #80                 1 - #85                 1 -    #90    - ATC CAG CAA GCA ATT TCC TAT TCC AGT TTT CC - #A TTC TGG ATG GGG CTG     744    Ile Gln Gln Ala Ile Ser Tyr Ser Ser Phe Pr - #o Phe Trp Met Gly Leu    #               205    - TCT CGG AGG AAC CCC AGC TAC CCA TGG CTC TG - #G GAG GAC GGT TCT CCT     792    Ser Arg Arg Asn Pro Ser Tyr Pro Trp Leu Tr - #p Glu Asp Gly Ser Pro    #           220    - TTG ATG CCC CAC TTA TTT AGA GTC CGA GGC GC - #T GTC TCC CAG ACA TAC     840    Leu Met Pro His Leu Phe Arg Val Arg Gly Al - #a Val Ser Gln Thr Tyr    #       235    - CCT TCA GGT ACC TGT GCA TAT ATA CAA CGA GG - #A GCT GTT TAT GCG GAA     888    Pro Ser Gly Thr Cys Ala Tyr Ile Gln Arg Gl - #y Ala Val Tyr Ala Glu    #   250    - AAC TGC ATT TTA GCT GCC TTC AGT ATA TGT CA - #G AAG AAG GCA AAC CTA     936    Asn Cys Ile Leu Ala Ala Phe Ser Ile Cys Gl - #n Lys Lys Ala Asn Leu    255                 2 - #60                 2 - #65                 2 -    #70    - AGA GCA CAG TGA ATTTGAAGGC TCTGGAAGAA AAGAAAAAAG TC - #TTTGAGTT     988    Arg Ala Gln  *    - TTATTCTGGA ATTTAAGCTA TTCTTTGTCA CTTGGGTGCC AAACATGAGA GC - #CCAGAAAA    1048    - CTGTCATTTA GCTGGCTGCA GAACTCCTTT GCAGAAACTG GGGTTCCAGG TG - #CCTGGCAC    1108    - CTTTATGTCA ACATTTTTGA TTCTAGCTAT CTGTATTATT TCACCTAGCT TG - #TCCCAAGC    1168    - TTCCCTGCCA GCCTGAAGTC CATTTTCCCC TTTTTATTTT AAAATTTGAC TC - #CTCTTCAA    1228    - GCTTGAAAAC CCTCTGAACT CAGTCTTCTT TACCTCATTA TCACCTTCCC CT - #CACACTCC    1288    #         1318     ACAG ACCGGAATTC    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  273 ami - #no acids              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - Met Thr Phe Asp Asp Leu Lys Ile Gln Thr Va - #l Lys Asp Gln Pro Asp    #                 15    - Glu Lys Ser Asn Gly Lys Lys Ala Lys Gly Le - #u Gln Phe Leu Tyr Ser    #             30    - Pro Trp Trp Cys Leu Ala Ala Ala Thr Leu Gl - #y Val Leu Cys Leu Gly    #         45    - Leu Val Val Thr Ile Met Val Leu Gly Met Gl - #n Leu Ser Gln Val Ser    #     60    - Asp Leu Leu Thr Gln Glu Gln Ala Asn Leu Th - #r His Gln Lys Lys Lys    # 80    - Leu Glu Gly Gln Ile Ser Ala Arg Gln Gln Al - #a Glu Glu Ala Ser Gln    #                 95    - Glu Ser Glu Asn Glu Leu Lys Glu Met Ile Gl - #u Thr Leu Ala Arg Lys    #           110    - Leu Asn Glu Lys Ser Lys Glu Gln Met Glu Le - #u His His Gln Asn Leu    #       125    - Asn Leu Gln Glu Thr Leu Lys Arg Val Ala As - #n Cys Ser Ala Pro Cys    #   140    - Pro Gln Asp Trp Ile Trp His Gly Glu Asn Cy - #s Tyr Leu Phe Ser Ser    145                 1 - #50                 1 - #55                 1 -    #60    - Gly Ser Phe Asn Trp Glu Lys Ser Gln Glu Ly - #s Cys Leu Ser Leu Asp    #               175    - Ala Lys Leu Leu Lys Ile Asn Ser Thr Ala As - #p Leu Asp Phe Ile Gln    #           190    - Gln Ala Ile Ser Tyr Ser Ser Phe Pro Phe Tr - #p Met Gly Leu Ser Arg    #       205    - Arg Asn Pro Ser Tyr Pro Trp Leu Trp Glu As - #p Gly Ser Pro Leu Met    #   220    - Pro His Leu Phe Arg Val Arg Gly Ala Val Se - #r Gln Thr Tyr Pro Ser    225                 2 - #30                 2 - #35                 2 -    #40    - Gly Thr Cys Ala Tyr Ile Gln Arg Gly Ala Va - #l Tyr Ala Glu Asn Cys    #               255    - Ile Leu Ala Ala Phe Ser Ile Cys Gln Lys Ly - #s Ala Asn Leu Arg Ala    #           270    - Gln    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 28 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    #= "PCR primer"ESCRIPTION: /desc    -    (iii) HYPOTHETICAL: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    #             28   CAAT AGATTCGC    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 27 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    #= "PCR primer"ESCRIPTION: /desc    -    (iii) HYPOTHETICAL: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    #             27   TAAA GAAACAG    __________________________________________________________________________

We claim:
 1. An isolated DNA sequence comprising a DNA sequence encodinga mammalian vascular endothelial receptor for modified low-densitylipoprotein, said sequence being selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, a DNA sequence encoding SEQ IDNO: 2, a DNA sequence encoding SEQ ID NO: 4, and a DNA sequence encodingSEQ ID NO:
 6. 2. An isolated DNA sequence encoding an amino acidsequence of amino acid Nos. 140 to 270 in SEQ ID NO: 2 SEQ ID NO: 4 orSEQ ID NO:
 6. 3. An isolated DNA sequence encoding a mammalian vascularendothelial receptor for modified low-density lipoprotein, said sequencebeing set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:
 5. 4. Anisolated DNA sequence comprising a recombinant transcriptional unitcomprising inducible regulatory expression elements derived from amicrobial or viral operon operably linked to a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, a DNAsequence encoding SEQ ID NO: 2, a DNA sequence encoding SEQ ID NO: 4,and a DNA sequence encoding SEQ ID NO:
 6. 5. A process for production ofa vascular endothelial receptor for modified low-density lipoprotein,which comprises transfecting the recombinant transcriptional unit ofclaim into a host cell and culturing the cell under conditions promotingexpression.
 6. A recombinant expression vector which contains a DNAsequence encoding a mammalian vascular endothelial receptor for modifiedlow-density lipoprotein, said sequence being set forth in SEQ ID NO: 1,SEQ ID NO: 3 or SEQ ID NO:
 5. 7. A purified protein compositioncontaining a vascular endothelial receptor for modified low-densitylipoprotein which is produced by a recombinant cell culture, saidreceptor having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO
 6. 8. A purifiedpeptide comprising an amino acid sequence of amino acid Nos. 140 to 270in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:
 6. 9. An isolated agent fordetecting modified low-density lipoprotein, wherein the agent comprisesa vascular endothelial receptor for modified low-density lipoprotein,said receptor having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO 6.